Sealing a growing brain

The brain of a human or another vertebrate begins in the early embryo as a simple tube made of cells. As it develops, it expands like a shaped balloon that is pinched off in some regions and stretches in others, creating bulbs and finger-like cavities called ventricles. The inside isn't filled with air but with cerebrospinal fluid, and scientists have wondered what prevents it from leaking through gaps between cells during this early stage in which it expands. Salim Seyfried's group at the MDC, collaborating with scientists from the FMP, has now found part of the answer: A protein that helps seal off the adult brain, protecting it from infections and toxins, also helps seal the neural tube during early stages of development. Their work, which appears in the January 26 edition of the journal PNAS, may help scientists find new ways to deliver drugs or other substances to the brain.

Two stages of the development of a zebrafish brain, showing claudin5a (blue). Top: within just a few hours of fertilization of the zebrafish egg, and prior to the formation of brain ventricles, claudin5a is strongly expressed within the developing central nervous system. This includes the hindbrain (hb) and spinal cord (sc). Weaker expression can be seen within the dorsal midbrain region (mb). Bottom: During ventricle expansion, several hours later, claudin5a is strongly expressed within the spinal cord and the neuroepithelial ventricular zones of the ventral hindbrain and midbrain. There is also strong expression in the forebrain ventricular zone. 

Recently scientists discovered that the neural tube grows properly with the help of molecules that regulate the way tissues absorb water. This supports a balloon model in which the tube grows because of pressure created by the fluid inside. Preventing leaks is just as important as filling the tube; otherwise, the early brain doesn't achieve its proper shape and fails to develop properly. PhD student Jingjing Zhang and other members of Salim's lab decided to look for the molecular seals. They worked with a tiny model organism called the zebrafish, which is virtually transparent, permitting them to watch developmental processes in a living animal.

Their starting point was the fact that in later stages of development, the brain is protected through a system called the blood-brain barrier. Its blood vessels are sealed more tightly than other parts of the body, preventing bacteria and fluids from leaving the bloodstream and entering the brain tissue. Proteins called Claudins latch onto each other to help form tight junctions between cells in the brain's blood vessels, as well as the skin and other tissues which have to be sealed. Recently other labs have shown that Claudins prevent leakage in other parts of the body during embryonic development. Jingjing and his colleagues wondered if they might be doing the same thing in the neural tube.

They discovered that the lining of the tube contains high amounts of one member of this protein family, called Claudin5a. The scientists "knocked-out" the claudin5a gene in early zebrafish embryos and checked for leaks by injecting the neural tube with a substance that could be seen under the electron microscope. They discovered that the tube failed to expand properly and that the substance leaked out into surrounding tissues. Removing Claudin5a didn't stop fluid from being pumped into the tube; instead, it just loosened the seals.

Claudin5a is a complex molecule with several regions, and Jingjing and his colleagues wanted to discover which part was acting as the seal. They generated zebrafish with an altered version of Claudin5a that had a defect in a specific region of the molecule and encountered a surprise: a module called the ECL2 domain was essential in stopping leaks. While studies of other tissues had shown that other parts of the protein help establish tight junctions, this was the first time that ECL2 has been shown to prevent leakage.

The close evolutionary relationship between fish and humans means that they have similar proteins; human versions of Claudin also have an ECL2 module that likely behaves in the same way. That could be important, Salim says, because there are cases in which you might want to unclamp the seals.

"The blood-brain barrier is an obstacle to delivering drugs or therapeutic substances to the brain," he says. "ECL2, and more generally Claudin5a, give us a new, specific target that you might be able to manipulate without affecting the integrity or structure of the tissue. You might be able to ease it open for short periods of time during a treatment. This work provides a model system – early zebrafish embryos – in which we can investigate such questions."

- Russ Hodge  

Highlight Reference:

Zhang J, Piontek J, Wolburg H, Piehl C, Liss M, Otten C, Christ A, Willnow TE, Blasig IE, Abdelilah-Seyfried S. Establishment of a neuroepithelial barrier by Claudin5a is essential for zebrafish brain ventricular lumen expansion. Proc Natl Acad Sci U S A. 2010 Jan 26;107(4):1425-30.

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